It has been observed that not only endotoxins of gram-negative bacteria but also some non-endotoxin components from these cells can stimulate bone resorption (BR) in an in vitro bone culture system. This proposal sets two major objectives to explore this observation in detail. The first objective is the isolation of the active bone resorbing component from Actinobacillus actinomycetemcomitans (Aa) strain Y4. Isolation will include all known procedures for extraction and fractionation of gram negative bacterial cell components. Purification procedures will include HPLC, TLC and ion-exchange chromatography as well as electrophoretic methods. The purification will be monitored by the Ca-45 labeled embryonic bone resorption assay. The purified and homogeneous BR stimulating components will be subjected to chemical structural analysis. The second objective of this project is to determine whether the BR stimulating factor has direct effects on both aspects of the bone remodeling cycle, bone resorption and bone formation. This will be approached by studies with isolated populations of bone forming and bone resorbing cells. In cells of the osteoblast line, we will test the BR stimulating factor and other components extracted from Aa for mitogenic activity and for effects on cell differentiation and function. In isolated osteoclast and macrophage preparations, we will test the BR components for prostaglandin production, calcified matrix removal from devitilized bone by both cells, and acid phosphatase production by isolated osteoclasts.